Rigorous and innovative studies of protein folding have broad implications to many areas of biological research and human health. Beyond aiding in the prediction of protein structure from sequence, the relevance of folding studies is underscored by the involvement of misfolded conformers in a variety of human diseases and the role of "natively-unfolded" proteins in regulation, recognition, and binding. Although considerable progress has been made in mechanistic studies, many fundamental issues remain. What types of structures compose the transition state ensemble? How well do experimental data agree with theoretical predictions, and what is the best way to conduct such comparisons? What are the structures of high energy intermediates? What are the unifying themes? Our long-term goal is to address these issues, thereby, generating a comprehensive description of how proteins adopt their native structures. In Aim 1, we will characterize folding transition states for a variety of proteins using our recently developed psi-analysis method. Psi-analysis is a major advance, that uniquely distinguishes fractional interactions and multiple pathways, identifies chain topologies; and provides site-resolved energetic information. We will characterize the transition state for a variety of proteins, to identify the prominence of structurally disjoint transition states (multiple pathways); evaluate theory and benchmark mutational phi- analysis studies by studying a "mu/sec folder", for which theoretical simulations and experiments have failed to provide a clear picture; and test our theory connecting native and transition state topologies. In Aim 2, we will determine the energy and structures of pre- and post-transition state "hidden intermediates" and test our predicted pathway for ubiquitin. In Aim 3, we will stabilize and characterize the structure of folding intermediates that form on the uphill side of the rate-limiting barrier, to obtain a nearly complete description of the folding pathway. Using hydrogen exchange and NMR methods, we will test our prediction that anomalously low mutational phi-values are due to structural relaxation in the transition state. The result is critical to the proper interpretation of numerous mutational folding and other biochemical studies.